Development of a soluble complex of I-Ek/d-moth cytochrome C (I-Ek/d- MCCO linked to a pentameric platform of cholera toxin-B (CT-B) subunits is proposed to develop immunoconjugates for direct detection of antigen- specific T cells. In preliminary work, the I-Ek/d-MCC construct has been successfully modified to include one-half of a leucine zipper and expressed in baculovirus-infected Sf9 insect cells. The cT-B-leucine zipper portion of the proposed complex is under development for expression in E. coli. Expressed proteins will be purified from culture supernatants by affinity and gel filtration chromatography and mixed to form the detection reagent. To demonstrate 'proof of principle', the resulting pentameric complex will be used to stain T cell lines specific for I-Ek/d-MCC with analysis by flow cytometry. We also propose to further study, optimize and develop additional MHC-peptide-CT-B complexes that will ultimately have utility in a clinical setting (e.g., isolation of tumor-specific CTLs for ex vivo expansion). It is anticipated that this novel platform will prove to be a more sensitive method for direct detection of antigen-specific T cells than technologies currently available. PROPOSED COMMERCIAL APPLICATIONS: There is a need for panels of reliable, sensitive commercial reagents for the detection of antigen-specific T cells in a variety of disease states (e.g., cancer, autoimmune disease). Although there are presently no commercial sources for these reagents, the potential market for MHC-peptide immunoconjugates is believed to be very large. The proposed platform technology is expected to provide a superior alternative to technologies currently employed in research labs worldwide.